The long-term goal is to develop a novel HBV antiviral strategy, that when used in combination with existing antiviral agents, will provide a more powerful therapy for treating chronically infected patients. Existing antivirals target reverse transcription. A second approach, but less effective and with significant side-effects, utilizes interferon therapy. Other approaches have suggested antisense, ribozymes, cytokines, and more recently siRNA. However, what should be a highly accessible target, virus attachment and entry, has been largely ignored, primarily due to lack of understanding as to how HBV actually attaches to and enters susceptible cells. With this deficit in mind, three Specific Aims are directed at important unsolved issues of virus assembly, attachment, entry, and the initiation of genome replication. These studies will exploit the fact that the assembly of human hepatitis delta virus (HDV), a natural satellite of human hepatitis B virus (HBV), is dependent upon HBV envelope proteins (HBsAg). There are already data to support the reasonable assumption that HBV and HDV use the same (or at least a very similar) mechanism for attachment and entry into susceptible cells. The strategy is to examine this mechanism exploiting the advantages of assaying HDV genome replication as the indicator for successful entry. The aims are as follows: (i) Establish conditions for the controlled assembly of HDV and evaluate in two different hepatocyte systems the ability to achieve attachment, entry, and the initiation of replication. (ii) Use unmodified and/or modified forms of HBV envelope proteins for virion assembly, and identify of determinants necessary for attachment, entry, and initiation of HDV replication. (iii) Test small molecules for interference with attachment and entry, and move towards the development of high throughput screening assays. Overall, the studies proposed in Specific Aims 1 and 2 provide new and important information relevant to how HBV (and HDV) are able to attach to and then enter and initiate replication in susceptible cells. This information in turn could lead in Specific Aim 3 to the development of inhibitors of infection at one or more steps. Then, in collaboration with Stressgen Biotechnologies, the developed assays will be used to screen multiple compounds to increase efficacy, and ultimately move to clinical studies.